Adhesion of cells to other cells and to the extracellularmatrix is a fundamental characteristicof all multicellular organisms. Adhesions providenot onlystructurallinksbetween the intracellularcytoskeletonand theextracellular environment, but also provide sites of signal transductionthat affect many aspects of cell behavior. This grant is aimed at understandinghow signals from cell-matrix and cell-cell adhesions regulate members of the Rho family of GTPases, which are themselves key regulators of the cytoskeleton and many intracellular processes. The goal of the first aim is to determine how adhesion to the extracellular matrix protein fibronectin stimulates RhoA activity. The respectiveroles of integrins and syndecan-4 will be investigated. We will explore the contribution of specific integrin type, density of expression and clustering on RhoA activation. Wewill test thehypothesis that some of syndecan-4's effects are mediated through activation of Rapl, another low molecular weight GTPase. Strategies for identifying and isolating guanine nucleotideexchange factors (GEFs) involved in RhoA activation will be used. We will explore signaling pathwaysthat regulate these GEFs. Cells can sense the physical state of the surface to which they adhere and we will test the hypothesis that isometric tension can stimulate RhoA activity. RhoA activity will be measuredusing biosensors expressed in singlecells and live cell imaging. We will determine whetherthe applicationof tension to singlecells locally activatesRhoA. The second aim is directedin part toward identifying the Racl GEFs that are activated in response to cadherin engagement. Inpreliminary work, we have discovered that many Rho family GEFs bind to PDZ domains. Proteins with PDZ domains are typically enriched in cell-cell junctions. We will determine whether the binding of GEFs to PDZ domains serves to recruit them to cell junctions and whether this interaction regulates their activity.